A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)γa as a pancreatic beta cell-specific biomarker
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Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.
We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.
After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na+–K+-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2γa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2γa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2γa and loss of insulin-positive cells.
We propose human FXYD2γa as a novel beta cell-specific biomarker.
KeywordsBeta cell biomarkers Diabetes Functional genomics Imaging Pancreatic beta cells
Beta cell mass
FXYD domain containing ion transport regulator 2
Massive parallel signal sequencing
Positron emission tomography
Transcripts per million
Vesicular monoamine transporter 2
Ludwig Institute for Cancer Research
Beta Cell Gene Bank
Both type 1 diabetes and type 2 diabetes are characterised by a reduced functional beta cell mass (BCM) [1, 2, 3]. Reduction of the BCM in type 1 diabetes is the consequence of a sustained autoimmune attack, while decreased beta cell mass in type 2 diabetes is less pronounced and probably secondary to prolonged metabolic stress [1, 2, 3]. The ability to measure the BCM prospectively in diabetic patients would be an important asset in understanding the natural history and pathogenesis of diabetes and would accelerate the development of new therapies for the disease. Unfortunately, there are no available tools for reliable and non-invasive in vivo BCM imaging . The major difficulties in imaging the BCM are that islets are small (50–300 µm), constitute only 1–2% of the total pancreatic mass and are scattered in the exocrine tissue . Pancreatic islets are composed of different cell types and beta cells have a common origin with other islet endocrine cells, acinar cells and the pancreatic ductal cells, making it difficult to find beta cell-specific biomarkers.
In vivo imaging of the BCM will require a combination of high sensitivity and specificity. Positron emission tomography (PET) and single photon emission computed tomography have high sensitivity and do not require ex vivo labelling. Imaging is achieved using either islet-specific binding compounds or by the use of radioactively labelled compounds specifically taken up by transporters in the beta cells. Ideally, two basic criteria should be met for adequate beta cell imaging : (1) beta cells should retain the tracer 100- to 1,000-fold more than the exocrine cells; and (2) agents that label beta cell surface proteins should do this at high enough concentrations to overcome imaging signals arising from unbound tracer retained in the extracellular and vascular spaces, and from tracer bound to the other tissues surrounding the pancreas. Agents being tested currently for BCM imaging include glibenclamide , glucagon-like peptide 1 receptor [7, 8], sulfonylurea receptor , vesicular monoamine transporter 2 (VMAT2) [9, 10, 11, 12, 13] and gangliosides . Unfortunately, the uptake/binding of these agents to beta cells, compared with exocrine pancreas and non-beta cells, is insufficient to allow reliable imaging of the BCM [6, 15]. For instance, the most promising beta cell marker, VMAT2, is produced in beta cells and monoaminergic neurons  but recent studies indicate that VMAT2 is also present in other islet cells besides beta cells . Promising preliminary data were obtained with 11C-labelled dihydrotetrabenazine (the PET ligand binding specifically to VMAT2) in streptozotocin (STZ)-induced diabetic Lewis rats  and in the BB-DP rat , but the data obtained in baboons  and in patients with longstanding type 1 diabetes  are not conclusive. A recent study using an 18F-labelled epoxide derivative of tetrabenazine showed good pancreas specificity and significantly reduced uptake in rat liver , but it remains unclear whether substituting isotopes will produce better biodistribution results in larger mammals. As a whole, these observations emphasise the need for the identification of new beta cell biomarkers.
Here we used a systems biology approach on human islet massive parallel signal sequencing (MPSS) data sets and Affymetrix microarray data sets obtained in purified primary rat islet beta cells and non-beta cells. These studies identified FXYD domain containing ion transport regulator 2 (FXYD2)γa, a regulatory subunit of the Na+–K+-ATPase [17, 18, 19], as a novel and specific beta cell membrane protein.
Human tissue samples were obtained with institutional review board approval from the Erasme Hospital (Université Libre de Bruxelles, Belgium), the University Hospital Dijkzigt (University Medical Center Rotterdam, The Netherlands) and the Churchill Hospital (Oxford, UK). Pancreas tissue sections from three patients with type 1 diabetes (time since diagnosis of type 1 diabetes, 31 years, 5 years and 3 days respectively; age at time of death, 62, 22 and 7 years) were obtained through the Oxford Centre for Diabetes (Churchill Hospital). Human fetal pancreas tissue samples (15, 18, 19, 24 and 25 weeks) were kindly provided by R. de Krijger (University Medical Center Rotterdam) to the Cell Differentiation Unit, Diabetes Research Center (Vrije Universiteit Brussel, Brussels, Belgium).
Pancreas sections from adult macaques (Macacus cynomolgus) treated or untreated with the diabetogenic drug STZ were obtained from the Laboratory of Experimental Surgery (Université Catholique de Louvain, Louvain-la-Neuve, Belgium). To induce diabetes (defined as a fasting blood glucose concentration >8.3 mmol/l), a low dose of filter-sterilised STZ (50 mg/kg body weight; Sigma, Bornem, Belgium) was administered intravenously over 5 min through the jugular vein . One month after STZ injection, STZ-treated animals had significantly higher values for fasting blood glucose concentration (12.0 ± 2.7 vs 3.3 ± 0.5 mmol/l, p < 0.005), polyuria (609 ± 138 vs 90 ± 18 ml/24 h, p < 0.005), polydipsia (1,167 ± 147 vs 342 ± 17 ml/24 h, p < 0.005) and glycosuria (10 g/l vs no glucose) compared with animals not treated with STZ .
Animal care and welfare
All animal experiments were performed in accordance with accepted principles of good laboratory animal care and according to protocols approved by the Université Libre de Bruxelles (rodent experiments) and Université Catholique de Louvain (primate studies) Animal Care and Use Committees.
Screening of beta cell-specific biomarkers
Massively parallel signature sequencing (MPSS) data sets of two human islet preparations from healthy donors  were compared with a published data set of 32 normal human tissues  (complete data set available from Ludwig Institute for Cancer Research, http://mpss.licr.org). Mean signature counts for specific genes in islet samples were compared with counts for the same genes in other tissues: signatures that differed between islets and the other tissues by at least 4 SD of the mean and with a minimum count of 50 transcripts per million (tpm) were retained as ‘pancreatic islet-enriched genes’. This list of 883 genes enriched in pancreatic islets was introduced into TMHMM  and Ingenuity Pathway Analysis (IPA) 5.0 [24, 25] to select genes encoding membrane proteins. In parallel, Affymetrix (Santa Clara, CA, USA) microarray data obtained in primary FACS-purified rat islet beta cells and non-beta cells [21, 26, 27, 28] were compared to obtain selected beta cell-specific genes encoding membrane proteins.
FXYD2 splice variants produced in pancreatic islets
Poly(A) + RNA was isolated from mouse and human pancreatic islets using oligo(dT)-coated polystyrene Dynabeads (Dynal, Oslo, Norway), and reverse-transcribed as previously described [32, 33]. All primers  are described in the Electronic supplementary material (ESM).
Monoclonal antibody and development of polyclonal antibodies directed against tissue-restricted human splice variants
A monoclonal antibody directed against human FXYD2γb (1C3-B3) was purchased from Abnova (Taipei, Taiwan). Rabbit polyclonal antibodies were produced against the two islet-specific human membrane proteins of FXYD2 following the Super Speedy protocol (Eurogentec, Seraing Belgium). For this purpose, the following splice variant-specific peptides in the N-terminal extracellular domain of human FXYD2 were chosen: EP070936 (MTGLSMDGGGS) for FXYD2γa and EP070937 (MDRWYLGGS) for FXYD2γb. Two rabbits were injected per peptide and rabbit polyclonal antibodies were affinity-purified against the respective peptides. Using this approach, novel rabbit polyclonal antibodies directed against human FXYD2γa (SPY393) and FXYD2γb (SPY394), were developed.
Splice variant specificity was confirmed by ELISA and by western blotting, including testing with the respective blocking peptides (ESM Fig. 1b).
Total protein extracts were prepared from rat kidney and pancreatic islets, resolved on Tricine–SDS gels (15% polyacrylamide) and transferred to polyvinylidene fluoride membranes. SPY393 polyclonal antibody (1:1,000; Eurogentec) was used to detect FXYD2γa. The secondary antibody used was a horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:5,000; Amersham Life Sciences, Amersham, UK). Membranes were incubated with the detection system of enhanced chemiluminescence (ECL; Supersignal West Femto Maximum Sensitivity Substrate; Pierce, Rockford, IL, USA). The protein-specific signals were identified using Biomax 1D image analysis software (Eastman Kodak, Rochester, NY, USA).
Paraffin-embedded human adult tissues (3 µm) were analysed with either 0.5 µg/ml monoclonal 1C3-B3 anti-FXYD2 or 7.5 µg/ml polyclonal SPY393 antibodies after antigen retrieval in citrate buffer, as described in Supplementary methods in the ESM.
Tissue microarray (human and macaque)
A human tissue microarray (TMA) was constructed with tissues from 36 organs with normal histology (with an average of three cases per organ and four spots per case). A normal pancreas TMA was constructed with tissue from 27 patients, each with ten spots (160 of these spots contained pancreatic islets).
Ten tissues were used for the construction of the macaque TMA.
Quantification and statistical analysis
Sections of pancreas from non-diabetic patients were analysed by immunofluorescence. Positive staining for insulin, glucagon and FXYD2γa was quantified by three observers unaware of sample identity and colocalisation between insulin and FXYD2γa and between glucagon and FXYD2γa was scored.
Sections of pancreas from non-diabetic and STZ-treated macaques were stained for IHC with SPY393, insulin or glucagon antibodies. After identification with the avidin–biotin–peroxidase complex method, six islets per case were quantified. Quantification was done with Spot Browser V2e software (Alphelys, Plaisir, France). Three observers, unaware of sample identity, scored for the presence of insulin, glucagon and FXYD2γa.
A systems biology approach identifies 12 beta cell-specific membrane markers
Islet specificity criteria based on comparison of quantitative MPSS data from human islets against MPSS data from 32 human tissues. We used as the cut-off point >50-fold enrichment of expression in islets vs whole pancreas.
Beta cell specificity criteria, i.e. higher gene expression in islet beta than in alpha cells.
Expression levels should be higher than or comparable to glucokinase, an enzyme produced in beta cells at a ratio of 9 tpm in the MPSS data set.
Selected biomarkers should be located in the plasma membrane. We first used the TMHMM program to detect transmembrane regions and then selected plasma membrane proteins using the Ingenuity Pathway Analysis program, which was followed by a thorough literature search.
Expression levels of the selected markers should not be modified to any major extent by inflammation, as evaluated by our microarray data sets obtained in purified primary rat beta cells [26, 28, 30, 32] and in human islets .
Using this selection procedure (Fig. 1), we identified 12 beta cell-specific membrane proteins that were not significantly modified by cytokine treatment. These candidate biomarkers were further evaluated as described below. Nine of the 12 genes were not detected in total pancreas MPSS data, while all of them have expression levels higher than 88 tpm in human islets. Two genes (FXYD2, GNAS/XLAS and GNAS/ALEX—the last two genes are both annotated to GNAS loci in the MPSS data) showed 500-fold and 1,280-fold enrichment, respectively, in islet vs total pancreas MPSS data. After this step, four candidates (ESM Table 1) were selected for further validation at the protein level regarding islet specificity in the human pancreas; this was done by immunohistochemistry and by analysis of human TMA (data not shown for XLAS, ALEX and annexin 7 [ANXA7]). FXYD2 was selected as the best candidate and only results regarding FXYD2 are presented in this paper.
Validation of the candidate biomarker in a human tissue microarray
Human FXYD2γa is identified as a beta cell-specific protein
Three splice variants of FXYD2 have been reported in mouse kidney, namely FXYD2γa, -γb and -γc, differing only in the aminoterminal extracellular sequence. Using mouse Fxyd2 splice-specific PCR primers , we identified two of the three splice variants in mouse islets; namely, a fragment of 223 bp (Fxyd2γa) and a fragment of 211 bp (Fxyd2γb), while no fragment of 343 bp (Fxyd2γc) was detected (ESM Fig. 1a). Similar data were obtained using splice-specific PCR primers for the human FXYD2 in three different human islet preparations; namely, a fragment of 198 bp (FXYD2γa) and a fragment of 192 bp (FXYD2γb), but no fragment of 325 bp (FXYD2γc) (ESM Fig. 2a).
To further characterise the presence of these spliced variants, specific antibodies directed against the first 11 amino acids from the aminoterminal extracellular domain of FXYD2γa (SPY393; Eurogentec) and FXYD2γb (SPY394; Eurogentec) were produced. Both isoforms were detected as membrane and intracellular proteins in INS-1E cells and primary rat beta cells (data not shown). In western blotting, the antibody directed against the γa isoform (SPY393) detected a 20 kDa band in rat pancreatic islets (ESM Fig. 1b, lane 5) and kidney (ESM Fig. 1b, lanes 1, 2 and 6). The human FXYD2γa peptide, but not the FXYD2γb peptide, specifically blocked detection of this band by SPY393 (ESM Fig. 1b, lanes 3 and 4 and ESM Fig. 1b, lanes 5 and 6). In human TMA, FXYD2γa was restricted to the pancreas, while FXYD2γb was expressed both in the pancreas and in the kidney tubules (ESM Table 2). Therefore, further validation was performed only with the SPY393 antibody, which recognises the γa isoform.
Exposure of human islets or rat dispersed islet cells for 24 h to cytokines (IL1β + IFNγ) did not change the expression of FXYD2γa at either the mRNA or the protein level (ESM Fig. 2).
Decreased FXYD2γa in islets from patients with type 1 diabetes
FXYD2γa and insulin decreased in parallel in macaques treated with streptozotocin
Percentage of cells staining for insulin, glucagon and FXYD2γa in pancreas sections from STZ-treated and untreated control macaques
Beta cells as percentage of total pancreas cells
Positive cells (%)a
54.9 ± 13.1
55.1 ± 8.1
59.5 ± 8.7
12.4 ± 6.4
68.3 ± 7.5
14.3 ± 7.0
23.4 ± 11.1
68.1 ± 3.3
10.6 ± 4.0
7.2 ± 6.5
80.1 ± 10.1
2.3 ± 2.0
22.0 ± 13.7
68.3 ± 7.1
11.3 ± 9.5
12.4 ± 6.4
69.6 ± 4.3
4.5 ± 0.6
12.6 ± 4.9
72.0 ± 10.4
4.0 ± 3.0
We have developed a functional genomics-based strategy to identify beta cell-specific biomarkers with potential for non-invasive imaging of the BCM. For this purpose, we compared a unique human islet MPSS data set  against MPSS data sets obtained in 32 human tissues . This allowed the identification of 114 islet-specific membrane genes with a minimum count of 50 tpm. The quantitative MPSS data used in the present study enabled us to calculate enrichment in human islets vs total pancreas. The mRNAs with the highest level of enrichment identified in this study (FXYD2, GNAS/XLAS, GNAS/ALEX and ANXA7; ESM Table 1 and data not shown) were selected for validation in human pancreas by immunohistochemistry and tested in a human tissue microarray containing 36 human tissues.
The best candidate beta cell biomarker identified, FXYD2, is a regulating subunit of the Na+–K+-ATPase. It was found to be 500-fold enriched in pancreatic islets compared with total pancreas. This gene has not been reported previously as being expressed in pancreatic islets. Using PCR, we confirmed that two out of the three known splice variants of FXYD2  are expressed in human pancreatic islets, and then produced polyclonal antibodies targeting specifically these human splice variant proteins for subsequent studies. Histological examination of human pancreas confirmed the islet-specific localisation of FXYD2γa and FXYD2γb. These two splice variants were previously reported as being produced in kidney proximal tubules and in distal convoluted tubules respectively [17, 18]. As the present data indicate, the FXYD2γb subunit is present in both kidney and pancreatic islets, while FXYD2γa is only present in human pancreatic islets. Importantly, FXYD2γa is mostly produced in beta cells, but not in alpha, delta or pancreatic polypeptide cells. Analysis of human fetal samples indicates that FXYD2γa expression is an early embryonic event, which may precede insulin expression. On the other hand, tumoral transformation of beta cells in human insulinomas is paralleled by loss of FXYD2γa expression.
A set of 35 human islet membrane-associated proteins were previously identified in an elegant study by Maffei et al.  using Affymetrix oligonucleotide chips to compare expression profiles of human islets against exocrine pancreas, liver and kidney. Most of the membrane genes identified in that study were also present in our list. Of note, this previous study  detected neither FXYD2, the most promising marker so far identified, nor GNAS/ALEX or GNAS/XLAS. The probable reason, at least for FXYD2, is that the probe sets for this mRNA in the Affymetrix microarrays do not provide consistent results (D. Flamez and D. L. Eizirik, unpublished data).
In order to quantify the remaining BCM in type 1 diabetes, markers selected for imaging should not be present in the infiltrating mononuclear cells and there should be a good correlation between beta cell loss and disappearance of the marker. FXYD2 expression was not detected in lymphocytes or other immune cells in the Symatlas data set  and FXYD2γa expression was not significantly modified by cytokines at either the mRNA or the protein level. Furthermore, there were clear and parallel decreases in FXYD2γa and insulin staining in pancreas both from individuals with type 1 diabetes and from macaques treated with STZ.
As mentioned above, FXYD2γa is a regulating subunit of the ubiquitous protein Na+–K+-ATPase. Na+-K+-ATPase has two obligatory subunits, a catalytic α subunit and a glycoprotein β subunit. Regulation of this pump occurs at several levels: gene expression, recruitment and internalisation of active pump subunits to/from the plasma membrane, and modulation of ATPase activity. The intrinsic properties of the pump are regulated by association with tissue-specific small single-span membrane proteins belonging to the FXYD family. A human FXYD2 G41R mutation is responsible for renal hypomagnesaemia , while silencing and overexpression of the γ subunit of Na+-K+-ATPase affects survival of mouse kidney (IMCD3) cells exposed to hypertonic stress .
A recent study shows the association of hepatocyte nuclear factor 1-β (HNF1β) with renal hypomagnesaemia and suggests that HNF1β regulates the transcription of FXYD2 . Little is known about the function of FXYD2γa and FXYD2γb in beta cells and future experiments, involving overexpression and knockdown of FXYD2γa and FXYD2γb in beta cells, should be performed to clarify their role in cell function and survival.
In conclusion, we have developed a systems biology approach, starting from MPSS and microarray data, for the identification of novel beta cell biomarkers. This, coupled with the systematic use of human tissue microarrays, allowed the identification of 12 proteins that are enriched in beta cells and have the potential to serve as biomarkers. Of special interest, the splice variant FXYD2γa was identified as beta cell-specific in both the human and a non-human primate model. This protein is not present in other islet cells, and its production decreases in parallel with beta cell loss in both human type 1 diabetes and in macaques rendered diabetic by injection of STZ. We propose that this newly identified beta cell protein, FXYD2γa, may be a useful biomarker for future non-invasive imaging and quantification of the pancreatic beta cell mass.
We thank M. A. Neef, M. Urbain and J. Schoonheydt of the Laboratory of Experimental Medicine (ULB) for excellent technical support. We also thank A. Verrellen and S. Kalista (Laboratory of Pathology, Erasme Hospital) for their assistance in the quantification analysis of the immunohistochemistry data. This work was supported by grants from the Pôles de compétitivité de la région Wallonne as part of the KeyMarker–BioWin programme (BioWin Convention No. 5437), European Union (BetaImage, Health-F2-2008-222980; in the Framework Programme 7 of the European Community), Fonds National de la Recherche Scientifique (FNRS) Belgium, the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State (IUAP P6/40), Juvenile Diabetes Research Foundation (JDRF) programme project (4-2001-438) and JDRF Bridge Support for Systems Biology of the Beta Cell (4-2008-376). B.K. is a recipient of a JDRF Postdoctoral Fellowship (3-2006-306).
Duality of interest
The biomarker described in this article is the subject of a patent deposited by Université Libre de Bruxelles (ULB), the Institute for Systems Biology (ISB) and Eurogentec SA.